A cornerstone of personalized cancer care will be the ability to predict how an individual patient will respond to a therapeutic intervention. Recent reports suggest that gene expression profiles have the potential to discriminate between patients who will and will not respond to specific chemotherapeutic agents. However, most existing gene expression predictive signatures have been developed and tested in fresh-frozen (FF) tissues and their utility in formalin fixed paraffin embedded (FFPE) samples, commonly encountered in clinical practice, is unknown.
Formalin fixation and wax embedding is a universal tissue processing procedure, allowing samples to be cut into thin sections (i.e. a few microns) stored at room temperatures indefinitely. Formalin-fixed paraffin-embedded (FFPE) archival clinical specimens are invaluable in discovery of prognostic and therapeutic targets for diseases such as cancer. Acquisition of appropriate clinical samples remains a fundamental problem in diagnosis. Tissue biopsies are difficult to obtain and therefore are too valuable to be used in global diagnosis development in most instances. A vast archive of tissue samples exists for every conceivable condition as formalin-fixed (FF) and paraffin-embedded (PE) samples. FFPE samples are prepared by incubating the tissue in a buffered formalin solution of 3.7% (w/v) formaldehyde and 10-15% methanol, forming intra- and inter-molecular covalent crosslinks between proteins, RNA and DNA (Fox, C. H., et al., Formaldehyde fixation. J. Histochem. Cytochem. 1985; 33:845-53; Kunkel, G. R., et al., Contact-site cross-linking agents. Mol. Cell. Biochem. 1981; 34:3-13). Afterwards, the samples are embedded with paraffin, which enables FFPE samples have been used to diagnose and stage tumors and evaluate protein expression by immunohistochemistry (IHC) and in situ hybridization.
Throughout a century of use, numerous archival paraffin-embedded tissue banks have been established worldwide. These tissue banks are invaluable resources of tissues for translational studies of cancer and various other diseases. Accessibility of macromolecules in the samples is a critical issue, as FFPE samples are traditionally limited to IHC.
Recent developments in extraction methodologies have opened FFPE samples to new analyses, like MS. An antigen retrieval (AR) technique, by boiling FFPE samples in water was shown to enhance IHC by circumventing the formalin fixation, and is now the typical approach for IHC staining of FFPE samples (Shi et al. 1991). Recently, AR and proteinase K/SDS treatment has been shown useful in extracting nucleic acids (Hood et al. 2006; Dubeau et al. 1986). These techniques rely on either strong heating of FFPE samples or enzyme digestion.
Proteomic studies of FFPE samples have been severely limited due to the formaldehyde-induced crosslinking, which renders proteins insoluble and unsuitable for biochemical extraction and analysis. For example, crosslinking prevents extraction of proteins from FFPE samples for use in protein analysis, such as Western blots. The advances in FFPE processing techniques have yet to overcome these obstacles, since many proteins are still undetectable (Crockett, D., et al., Identification of proteins from formalin-fixed paraffin-embedded cells by LC-MS/MS. Lab. Invest., 2005; 85:1405-1415).
A predictor for ovarian cancer response to platinum-based therapy is needed for use with stable patient samples, such as formalin fixed paraffin embedded samples.